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Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing of Gram-negative bacteria. However, any MD2 protein segment involved in phagocytosis of Gram-negative bacteria is not yet known. A short synthetic MD2 segment, MD54 (amino acid regions 54 to 69), was shown to interact with a Gram-negative bacterial outer membrane component, LPS, earlier. Furthermore, the MD54 peptide induced aggregation of LPS and facilitated its internalization in THP-1 cells. Currently, it has been investigated if MD2-derived MD54 possesses any opsonic property and role in phagocytosis of Gram-negative bacteria. Remarkably, we observed that MD54 facilitated agglutination of Gram-negative bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC BAA-427), but not of Gram-positive bacteria, Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923). The MD54-opsonized Gram-negative bacteria internalized within PMA-treated THP-1 cells and were killed over a longer incubation period. However, both internalization and intracellular killing of the MD54-opsonized Gram-negative bacteria within THP-1 phagocytes were appreciably inhibited in the presence of a phagocytosis inhibitor, cytochalasin D. Furthermore, MD54 facilitated the clearance of Gram-negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC BAA-427) from the infected BALB/c mice whereas an MD54 analog, MMD54, was inactive. Overall, for the first time, the results revealed that a short MD2-derived peptide can specifically agglutinate Gram-negative bacteria, act as an opsonin for these bacteria, and facilitate their phagocytosis by THP-1 phagocytes. The results suggest that the MD54 segment could have a crucial role in MD2-mediated host-pathogen interaction involving the Gram-negative bacteria.

The upper respiratory tract (nasopharynx or NP) is the first site of influenza replication, allowing the virus to disseminate to the lower respiratory tract or promoting community transmission. The host response in the NP regulates an intricate balance between viral control and tissue pathology. The hyper-inflammatory responses promote epithelial injury, allowing for increased viral dissemination and susceptibility to secondary bacterial infections. However, the pathologic contributors to influenza upper respiratory tissue pathology are incompletely understood. In this study, we investigated the role of interleukin IL-17 recetor A (IL-17RA) as a modulator of influenza host response and inflammation in the upper respiratory tract. We used a combined experimental approach involving IL-17RA-/- mice and an air-liquid interface (ALI) epithelial culture model to investigate the role of IL-17 response in epithelial inflammation, barrier function, and tissue pathology. Our data show that IL-17RA-/- mice exhibited significantly reduced neutrophilia, epithelial injury, and viral load. The reduced NP inflammation and epithelial injury in IL-17RA-/- mice correlated with increased resistance against co-infection by Streptococcus pneumoniae (Spn). IL-17A treatment, while potentiating the apoptosis of IAV-infected epithelial cells, caused bystander cell death and disrupted the barrier function in ALI epithelial model, supporting the in vivo findings., Competing Interests: All authors have read and approved the final version of the manuscript. The authors have declared that no conflict of interest exists., (Copyright: © 2023 Navaeiseddighi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study aims to redefine obesity cut-off points for body mass index (BMI) and fat mass index (FMI) according to the different age groups of physically active males. Healthy physically active volunteers (N = 1442) aged 18-57 years (y), with a mean BMI = 22.7 ± 2.8 kg/m 2 , and mean FMI = 4.3 ± 1.7 kg/m 2 were recruited from various fitness centers. BMI was calculated and individuals were categorized according to the Asia-Pacific BMI criterion of ≤22.9 kg/m 2 and the previous WHO-guided BMI criterion of ≤24.9 kg/m 2 . FMI was also calculated for the study participants with a cut-off of 6.6 kg/m 2 . Redefining of BMI and FMI cut-off values was carried out based on different age groups categorized with a difference of 10 y and 5 y using the receiver operating characteristic (ROC) curve and Youden's index. For the entire study population, BMI redefined cut-off points for overweight and obesity were 23.7 kg/m 2 and 24.5 kg/m 2 , respectively, while FMI redefined cut-off points for overweight and obesity were 4.6 kg/m 2 and 5.7 kg/m 2 , respectively. With 10 y of age group difference, a constant BMI and FMI values were observed, while with 5 y of age group difference, a constant increase in the BMI cut-offs was observed as the age group increased, i.e., from 23.3 kg/m 2 in 20-24 y to 26.6 kg/m 2 in ≥45 y and a similar trend was seen in FMI cut-offs. To conclude, our study suggests that age-dependent BMI and FMI cut-off points may provide appropriate measurements for physically active males as the age group increases.

Influenza A virus (IAV) infection triggers an exuberant host response that promotes acute lung injury. However, the host response factors that promote the development of a pathologic inflammatory response to IAV remain incompletely understood. In this study, we identify an interferon-γ (IFN-γ)-regulated subset of monocytes, CCR2 + monocytes, as a driver of lung damage during IAV infection. IFN-γ regulates the recruitment and inflammatory phenotype of CCR2 + monocytes, and mice deficient in CCR2 (CCR2 -/- ) or IFN-γ (IFN-γ -/- ) exhibit reduced lung inflammation, pathology, and disease severity. Adoptive transfer of wild-type (WT) (IFN-γR1 +/+ ) but not IFN-γR1 -/- CCR2 + monocytes restore the WT-like pathological phenotype of lung damage in IAV-infected CCR2 -/- mice. CD8 + T cells are the main source of IFN-γ in IAV-infected lungs. Collectively, our data highlight the requirement of IFN-γ signaling in the regulation of CCR2 + monocyte-mediated lung pathology during IAV infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophils are an important component of the innate immune response against various pathogens. However, there is a lack of research concerning the effects of short intensive training on neutrophil functions, especially neutrophil extracellular traps (NET) formation. The study aim was to determine the effects of a 19-day training cycle on innate immunity among young male athletes. Six male ice hockey players (< 20 years old) from the Polish national team were monitored across a five-day training camp and after a return to normal club training. The first blood collection took place before training (T1), the second after the training camp (T2) and the third 14 days later (T3). The counts/concentrations of blood biochemical, immune and endocrine markers were compared across each training period. Creatine kinase activity tended to increase at T2 (546 ± 216 U·L -1 ) when compared to T1 (191 ± 111 U·L -1 ; p=0.063). Neutrophil extracellular traps formation and neutrophil counts also differed between training periods (p=0.042 and p=0.042, respectively). Neutrophil counts tended to decrease, in contrast to NET formation which tended to rise, at T2 in comparison to T1 (2.51 ± 0.45 vs 3.04 ± 0.47 109·L -1 ; 24 ± 13 vs 8 ± 15%, respectively). No significant differences in other leucocyte counts were observed. A short period of intensive training was accompanied by some muscle damage and inflammation, as evidenced by CK and NET up-regulation, whilst neutrophil counts were diminished in the blood. Thus, neutrophils and NET could be involved in muscle damage and local inflammatory processes following intensive physical training in young male athletes., Competing Interests: The authors declare that they have no conflicts of interes., (Copyright © Biology of Sport 2021.)

Heat shock factor 1, HSF1, is one of several family members that recognize repeated nGAAn sequences associated with the heat shock element of heat shock and other genes. This transactivator is activated from a monomeric to trimeric form by oxidative, thermal and other stressors. Various studies show that HSF1 levels increase with cancer and decrease with aging and neurodegenerative disorders. It has a role in development as well as infections and inflammation. HSF1 is regulated by post-translational modifications and interactions with other proteins such as HSBP-1. Given its central importance in stress responsivity, various methods have been developed to identify HSF1 and its interacting partners. To date, multiple studies use conventional immunoprecipitation of HSF1 with commercially available antibodies which work well in cell lines but not whole tissue extracts. To remedy this shortfall, we developed a technique to retrieve activated HSF1 with an oligonucleotide link to a magnetic bead. The method captures HSF1 using a DNA sequence specific for HSF1 binding sites on promoter of heat shock genes. Confirmation of tissue derived HSF1 is identified using antibody against HSF1. The magnetic beads conjugated with DNA sequence specific to HSF1 binding was capable of yielding a reproducible band of high signal intensity with low background after native gel electrophoresis and ECL. Thus, the trimeric form of HSF1 can be isolated from tissue with magnetic beads conjugated with a short DNA sequence specific to HSF1 binding. This new method to identify HSF1 is economic, easy, and reproducible and does not require specialized equipment. It overcomes limitations of HSF1 tissue extraction by conventional immunoprecipitation, thus allowing for new approaches to understand HSF1 function in animal and human tissue.•HSF1 is a transcription factor that homotrimerize and binds to a conserved regulatory site, the heat shock element (HSE), consists of repeats of pentameric sequence '5-nGAAn-3' present in the promoters of inducible heat shock protein genes.•This protocol allows isolation of trimeric forms of HSF1 from tissue lysate using magnetic beads conjugated with a short DNA sequence with specific binding to HSF1.•This method is easy, economic and does not require unique instrumentation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

How the heat shock axis, repair pathways, and proteostasis impact the rate of aging is not fully understood. Recent reports indicate that normal aging leads to a 50% change in several regulatory elements of the heat shock axis. Most notably is the age-dependent enhancement of inhibitory signals associated with accumulated heat shock proteins and hyper-acetylation associated with marked attenuation of heat shock factor 1 (HSF1)-DNA binding activity. Because exceptional longevity is associated with increased resistance to stress, this study evaluated regulatory check points of the heat shock axis in liver extracts from 12 months and 24 months long-lived Ames dwarf mice and compared these findings with aging wild-type mice. This analysis showed that 12M dwarf and wild-type mice have comparable stress responses, whereas old dwarf mice, unlike old wild-type mice, preserve and enhance activating elements of the heat shock axis. Old dwarf mice thwart negative regulation of the heat shock axis typically observed in usual aging such as noted in HSF1 phosphorylation at Ser307 residue, acetylation within its DNA binding domain, and reduction in proteins that attenuate HSF1-DNA binding. Unlike usual aging, dwarf HSF1 protein and mRNA levels increase with age and further enhance by stress. Together these observations suggest that exceptional longevity is associated with compensatory and enhanced HSF1 regulation as an adaptation to age-dependent forces that otherwise downregulate the heat shock axis., (© 2021. The Author(s).)

Despite their protective antimicrobial function, neutrophil extracellular traps (NETs) have been implicated in propagation of inflammatory responses in several disease conditions including sepsis. Highly diffusible exogenous ROS produced under such inflammatory conditions, can induce exuberant NETs, thus making inhibition of NETs desirable in inflammatory diseases. Here we report that helminth parasite excretory/secretory factors termed as parasitic ligands (PL) inhibit ROS-induced NETs by blocking the activation of nonselective calcium permeable channel Transient Receptor Potential Melastatin 2 (TRPM2). Therapeutic implication of PL mediated blockage of NET formation was tested in preclinical model of septic peritonitis, where PL treatment regulated neutrophil cell death modalities including NET formation and mitigated neutrophil mediated inflammatory response. This translated into improved survival and reduced systemic and local bacterial load in infected mice. Overall, our results posit PL as an important biological regulator of neutrophil functions with implications to a variety of inflammatory diseases including peritonitis.

This study aims to identify the clinical and genetic markers related to the two uncommon nutritional statuses-metabolically unhealthy normal-weight (MUNW) and metabolically healthy overweight/obese (MHOW) individuals in the physically active individuals. Physically active male volunteers ( n = 120) were recruited, and plasma samples were analyzed for the clinical parameters. Triglycerides, HDL-Cholesterol, LDL-cholesterol, total cholesterol, C-reactive protein, and insulin resistance were considered as markers of metabolic syndrome. The subjects were classified as 'healthy' (0 metabolic abnormalities) or 'unhealthy' (≥1 metabolic abnormalities) in their respective BMI group with a cut-off at 24.9 kg/m 2 . Analysis of biochemical variables was done using enzyme linked immunosorbent assay (ELISA) kits with further confirmation using western blot analysis. The microarray was conducted, followed by quantitative real-time PCR to identify and analyze differentially expressed genes (DEGs). The MHOW group constituted 12.6%, while the MUNW group constituted 32.4% of the total study population. Pro-inflammatory markers like interleukin-6, tumor necrosis factor (TNF)-α, and ferritin were increased in metabolically unhealthy groups in comparison to metabolically healthy groups. Gene expression profiling of MUNW and MHOW individuals resulted in differential expression of 7470 and 5864 genes, respectively. The gene ontology (GO) biological pathway analysis showed significant enrichment of the 'JAK/STAT signaling pathway' in MUNW and 'The information-processing pathway at the IFN-β enhancer' pathway in MHOW. The G6PC3 gene has genetically emerged as a new distinct gene showing its involvement in insulin resistance. Biochemical, as well as genetic analysis, revealed that MUNW and MHOW are the transition state between healthy and obese individuals with simply having fewer metabolic abnormalities. Moreover, it is possible that the state of obesity is a biological adaptation to cope up with the unhealthy parameters.

Sepsis resulting from acute pneumonic infections by Gram-negative bacteria is often characterized by dysfunction of innate immune components. Here we report a previously unrecognized innate protective function of SAP, an adaptor protein primarily reported in T cells, NK cells, and NKT cells, during acute pneumonic infection with Klebsiella pneumoniae (KPn). SAP-deficient mice were highly susceptible to this infection with elevated systemic bacterial spread and increased lung damage. While the overall influx of infiltrating cells in the lungs remained largely intact, increased mortality of SAP-deficient mice correlated with increased accumulation of large NK1.1+ cells harboring bacteria and an impairment of neutrophil extracellular trap formation in vivo during KPn pneumonia, which likely facilitated bacterial outgrowth. Neutrophils were found to express SAP; however, adoptive transfer experiment supported a neutrophil-extrinsic function of SAP in neutrophil extracellular trap formation. Collectively, these data present the first report depicting innate protective function of SAP in an acute pulmonary infection., (Copyright © 2019. Published by Elsevier Ltd.)

Synergistic coexistence of nitrogen fixing cyanobacteria such as Anabaena variabilis, Nostoc muscorum and Westiellopsis prolifica with green algae namely Scenedesmus obliquus, Chlorella vulgaris and Botryococcus braunii was studied under nitrogen deficient conditions. The effect of these interactions was investigated on growth, fixed nitrogen content, lipid content and their secretomes in individual cultures and cocultures. Based on the cocultivation studies, it was found that out of the nine interactions studied, B. braunii-N. muscorum synergism was best established. This interaction resulted in a maximum of 50% enhancement in nitrogen fixation in B. braunii-N. muscorum co-culture leading to 27% enhancement in lipid content (membrane and neutral lipid). In general, B. braunii co-cultures showed an enhancement in biomass content of up to 38%. Secretome analysis showed presence of new and modified secondary metabolites having roles in quorum sensing/quenching, interspecies signaling, N-fixation, carbon metabolism, lipid metabolism, antimicrobial activity. Compounds such as trichloroacetic acid and hexadecane were identified that are known to have roles in nitrogen assimilation and carbon metabolism, respectively, were present in some of the co-culture secretomes. The combination of B. braunii-N. muscorum led to the formation of new compounds such as triacontanol which have role in improvement of glucose-lipid metabolism and 9-octadecenamide that is known to be a phytohormone., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Neutrophil extracellular trap (NET) formation constitutes an important extracellular antimicrobial function of neutrophils that plays a protective role in bacterial pneumonia. Formation of reactive oxygen species (ROS) such as highly diffusible hydrogen peroxide (H 2 O 2 ) is a hallmark of oxidative stress during inflammatory lung conditions including pneumonia. However, the impact of exogenous ROS on NET formation and the signaling pathway involved in the process is not completely understood. Here we demonstrate that the ROS-sensing, nonselective, calcium-permeable channel transient receptor potential melastatin 2 (TRPM2) is required for NET formation in response to exogenous H 2 O 2 . This TRPM2-dependent H 2 O 2 -mediated NET formation involved components of autophagy and activation of AMPK and p38 MAPK, but not PI3K and AKT. Primary neutrophils from Trpm2 - / mice were highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. These results identify a key role of TRPM2 in regulating NET formation by exogenous ROS via AMPK/p38 activation and autophagy machinery, as well as a protective antimicrobial role of TRPM2 in pneumonic bacterial infection.-Tripathi, J. K., Sharma, A., Sukumaran, P., Sun, Y., Mishra, B. B., Singh, B. B., Sharma, J. Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.- mice fail to activate this pathway with a block in NET release and a concomitant decrease in their antimicrobial capacity. Consequently, Trpm2 - / - mice were highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. These results identify a key role of TRPM2 in regulating NET formation by exogenous ROS via AMPK/p38 activation and autophagy machinery, as well as a protective antimicrobial role of TRPM2 in pneumonic bacterial infection.-Tripathi, J. K., Sharma, A., Sukumaran, P., Sun, Y., Mishra, B. B., Singh, B. B., Sharma, J. Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

Transient events that occur during plasma instabilities in fusion reactors impart large heat fluxes onto the surrounding plasma-facing components (PFCs). Erosion and splashing of PFCs can contaminate the plasma and shorten material lifetime. Although tungsten is currently considered the most promising candidate material for future PFCs, concerns over the thermal shock performance during type-I ELMs (transient events expected in fusion devices) necessitate the study of other comparable materials. ELM-like heat loading was applied via a pulsed Nd:YAG millisecond laser on a pristine molybdenum (Mo) surface to measure surface melting and mass loss. One potential advantage of Mo is its higher specific heat of vaporization, which could lead to reduced particle emission. Imaging of the surface after loading revealed that complete surface melting began at 1.0 MJ m -2 (heat load parameter of 31.62 MJ m -2  s -1/2 ). Photon excitation also increased significantly above 1.0 MJ m -2 , indicating possible phase change. At 1.4 MJ m -2 (44.27 MJ m -2 s -1/2 ), in situ mass loss measurements found an exponential increase in particle emission, indicating the presence of droplet formation and boiling. Direct comparisons of erosion during pulsed heat loading between PFC candidate materials will ensure that future fusion devices design components with optimal thermal strength.

The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni 2+ or Zn 2+ for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast-localized GLYI enzyme, OsGLYI-8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI-8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI-8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady-state kinetics with a low-affinity and a high-affinity substrate-binding component. Loss of AtGLYI-2, the closest Arabidopsis ortholog of OsGLYI-8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI-2 or OsGLYI-8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

The goal of this work is to assess Ta as a potential plasma-facing material for future fusion reactors in terms of its response to high-flux, low-energy He + ion irradiation. Ta samples were irradiated with 100 eV He + ions at various fluences up to 3.5 × 10 25  ions m -2 while simultaneously heated at constant temperatures in the range 823-1223 K. SEM studies show that irradiated Ta surfaces undergo significant morphology changes that have a strong dependence on both ion fluence and sample temperature. Optical reflectivity complements SEM and demonstrates a vertical growth of surface structures with increasing fluence. Ex situ XPS and XRD both show significant oxidation of the irradiated Ta surfaces, giving further qualitative information on the extent of surface modification. Overall, these irradiation-induced structures on Ta are similar to early-stage "fuzz" structures observed in W. However, Ta exhibits a higher fluence threshold for structure formation. While Ta may have less desirable bulk properties (e.g., thermal conductivity) when compared to W, its higher resilience to He + ion-induced surface modification suggests that surface thermal and mechanical properties may not degrade as quickly in extreme fusion environments; this quality may be a redeeming factor for Ta as a plasma-facing material.

We propose low-energy, broad-beam He + ion irradiation as a novel processing technique for the generation of Nb 2 O 5 surface nanostructures due to its relative simplicity and scalability in a commercial setting. Since there have been relatively few studies involving the interaction of high-fluence, low-energy He + ion irradiation and Nb (or its oxidized states), this systematic study explores both effects of fluence and sample temperature during irradiation on resulting surface morphology. Detailed normal and cross-sectional scanning electron microscopy (SEM) studies reveal subsurface He bubble formation and elucidate potential driving mechanisms for nanostructure evolution. A combination of specular optical reflectivity and X-ray photoelectron spectroscopy (XPS) is also used to gain additional information on roughness and stoichiometry of irradiated surfaces. Our investigations show significant surface modification for all tested irradiation conditions; the resulting surface structure size and geometry have a strong dependence on both sample temperature during irradiation and total ion fluence. Optical reflectivity measurements on irradiated surfaces demonstrate increased surface roughening with increasing ion fluence, and XPS shows higher oxidation levels for samples irradiated at lower temperatures, suggesting larger surface roughness and porosity. Overall, it was found that low-energy He + ion irradiation is an efficient processing technique for nanostructure formation, and surface structures are highly tunable by adjusting ion fluence and Nb 2 O 5 sample temperature during irradiation. These findings may have excellent potential applications for solar energy conversion through improved efficiency due to effective light absorption.

Piscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 heptad repeat., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

KLD-12 (KLD) is a 12-residue self-assembling peptide that can adopt nano-structures and is known for its tissue-engineering properties. Our objective was to introduce antimicrobial attribute to KLD which would help in preventing secondary infection associated with external application of such tissue engineering materials. Considering the net charge of KLD-12, varying number of cationic arginine residues were added to its N-terminus. KLD variants showed appreciable bactericidal properties without any significant increase in cytotoxicity against tested mammalian cells. Further, these variants adopted β-sheet structures and self-assembled into nano-structures comparable to that of KLD. Interestingly, the KLD variants with two (KLD-2R) and three (KLD-3R) arginine residues added to its N-terminus showed significant osteogenic effect which was comparable or better than the original peptide as evident from the alkaline phosphatase activity assay, mineralized nodule formation and expression of different osteogenic genes. Particularly, application of KLD-2R in rats to the site of a drill-hole (0.8 mm diameter) that was created in the femur metaphysis displayed significantly higher bone regeneration compared to that of KLD. The results demonstrate a simple way to improve biological property of a self-assembling peptide with tissue engineering property., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

IsCT, a 13-residue, non-cell-selective antimicrobial peptide is comprised of mostly hydrophobic residues and lesser cationic residues. Assuming that placement of an additional positive charge in the non-polar face of IsCT could reduce its hydrophobic interaction, resulting in its reduction of cytotoxicity, an analog, I9K-IsCT was designed. Two more analogs, namely, E7K-IsCT and E7K,I9K-IsCT, were designed to investigate the impact of positive charges in the polar face as well as polar and non-polar faces at a time. These amino acid substitutions resulted in a significant enhancement of therapeutic potential of IsCT. IsCT and E7K-IsCT seem to target bacterial membrane for their anti-bacterial activity. However, I9K-IsCT and E7K,I9K-IsCT inhibited nucleic acid and protein syntheses in tested E. coli without perturbing its membrane. This was further supported by the observation that NBD-IsCT localized onto bacterial membrane while NBD-labeled I9K-IsCT and E7K,I9K-IsCT translocated into bacterial cytoplasm. Interestingly, IsCT and E7K-IsCT were significantly helical while I9K-IsCT and E7K,I9K-IsCT were mostly unstructured with no helix content in presence of mammalian and bacterial membrane-mimetic lipid vesicles. Altogether, the results identify two novel cell-selective analogs of IsCT with new prototype amino acid sequences that can translocate into bacterial cytoplasm without any helical structure and inhibit macromolecular syntheses.

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at 'a' and/or 'd' position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its 'a' and 'd' positions with D-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its D-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at 'a' and/or 'd' positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.

The glyoxalase system constitutes the major pathway for the detoxification of metabolically produced cytotoxin methylglyoxal (MG) into a non-toxic metabolite D-lactate. Glyoxalase I (GLY I) is an evolutionarily conserved metalloenzyme requiring divalent metal ions for its activity: Zn(2+) in the case of eukaryotes or Ni(2+) for enzymes of prokaryotic origin. Plant GLY I proteins are part of a multimember family; however, not much is known about their physiological function, structure and metal dependency. In this study, we report a unique GLY I (OsGLYI-11.2) from Oryza sativa (rice) that requires Ni(2+) for its activity. Its biochemical, structural and functional characterization revealed it to be a monomeric enzyme, possessing a single Ni(2+) coordination site despite containing two GLY I domains. The requirement of Ni(2+) as a cofactor by an enzyme involved in cellular detoxification suggests an essential role for this otherwise toxic heavy metal in the stress response. Intriguingly, the expression of OsGLYI-11.2 was found to be highly substrate inducible, suggesting an important mode of regulation for its cellular levels. Heterologous expression of OsGLYI-11.2 in Escherichia coli and model plant Nicotiana tabacum (tobacco) resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lower Na(+) /K(+) ratio and maintenance of reduced glutathione levels. Together, our results suggest interesting links between MG cellular levels, its detoxification by GLY I, and Ni(2+) - the heavy metal cofactor of OsGLYI-11.2, in relation to stress response and adaptation in plants., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

FasL mediated preferential apoptosis of bystander CTLs while protection of infected CD4(+)T cells remains one of the hallmarks of immune evasion during HIV infection. The property of infected host cells to evade cell-autonomous apoptosis emanates from ability of HIV-1Nef-protein to physically interact with ASK-1 and thereby inhibit its enzymatic activity. The specific domains of HIV-1Nef through which it may interact with ASK1 and thereby impair the ASK1 activity remain unidentified so far and represent a major challenge towards developing clear understanding about the dynamics of this interaction. Using mammalian two hybrid screen in association with site directed mutagenesis and competitive inhibitor peptides, we identified constituent minimal essential domain (152 DEVGEANN 159) through which HIV-1Nef interacts with ASK1 and inhibits its function. Furthermore our study also unravels a novel alternate mechanism underlying HIV-1 Nef mediated ASK1 functional modulation, wherein by potentiating the inhibitory ser(967) phosphorylation of ASK1, HIV-1Nef negatively modulated ASK1 function.

To understand the influence of different hydrophobic amino acids at "a" and "d" positions of a heptad repeat sequence on antimicrobial, cytotoxic, and antiendotoxin properties, four 15-residue peptides with leucine (LRP), phenylalanine (FRP), valine (VRP), and alanine (ARP) residues at these positions were designed, synthesized, and characterized. Although valine is similarly hydrophobic to leucine and phenylalanine, VRP showed significantly lesser cytotoxicity than LRP and FRP; further, the replacement of leucines with valines at "a" and "d" positions of melittin-heptads drastically reduced its cytotoxicity. However, all four peptides exhibited significant antimicrobial activities that correlate well with their interactions with mammalian and bacterial cell membranes and the corresponding lipid vesicles. LRP most efficiently neutralized the LPS-induced pro-inflammatory mediators like NO, TNF-α, and IL-6 in macrophages followed by FRP, VRP, and ARP. The results could be useful for designing short antimicrobial and antiendotoxin peptides with understanding the basis of their activity.

Self-assembled α-FeSi(2) nanoislands were formed using solid-phase epitaxy of low (~1.2 ML) and high (~21 ML) Fe coverages onto vicinal Si(111) surfaces followed by thermal annealing. At a resulting low Fe-covered Si(111) surface, we observed in situ, by real-time scanning tunneling microscopy and surface electron diffraction, the entire sequence of Fe-silicide formation and transformation from the initially two-dimensional (2 × 2)-reconstructed layer at 300 °C into (2 × 2)-reconstructed nanoislands decorating the vicinal step-bunch edges in a self-ordered fashion at higher temperatures. In contrast, the silicide nanoislands at a high Fe-covered surface were noticeably larger, more three-dimensional, and randomly distributed all over the surface. Ex situ x-ray photoelectron spectroscopy and high-resolution transmission electron microscopy indicated the formation of an α-FeSi(2) island phase, in an α-FeSi(2){112} // Si{111} orientation. Superconducting quantum interference device magnetometry showed considerable superparamagnetism, with ~1.9 μ(B)/Fe atom at 4 K for the low Fe-coverage, indicating stronger ferromagnetic coupling of individual magnetic moments, as compared to high Fe-coverage, where the calculated moments were only ~0.8 μ(B)/Fe atom. Such anomalous magnetic behavior, particularly for the low Fe-coverage case, is radically different from the non-magnetic bulk α-FeSi(2) phase, and may open new pathways to high-density magnetic memory storage devices.

In the present study, storage proteins from five different wheat cultivars were extracted, fractionated and evaluated for their accumulation at different stages of development. SDS-PAGE analysis revealed that the accumulation of high molecular weight glutenin subunits was cultivar and stage dependent. However, low molecular weight glutenin subunits' accumulation was not altered significantly after 16days post anthesis in any of the cultivars. The rheological parameters (storage- and loss-modulus) of dough and gluten showed close association with either gliadins or glutenins. Peptidyl prolyl cis-trans isomerase (PPIase) activity, measured at different stages of grains development, showed variability with both the developmental stage and cultivar, and appeared to be primarily due to cyclophilins. Principal component analysis revealed the association of PPIase activity with either gliadin or total proteins, suggesting their significant role in the deposition of storage proteins in wheat., (Copyright © 2011 Elsevier Ltd. All rights reserved.)